Characterization of rhodopsin kinase purified from bovine rod outer segments.
Rhodopsin kinase was purified by sequential chromatography on DEAE-cellulose and blue-Sepharose. Kinase activity co-purified with a 62-kDa polypeptide, which bound light-dependently in the absence of ATP to purified vesicle-reconstituted rhodopsin. Purified rhodopsin kinase is free of any detectable arrestin or the retinal G-protein. Rhodopsin kinase is autophosphorylated on serine residues which is unaffected by the presence of bleached rhodopsin and results in a transition in molecular mass to 64 kDa. Autophosphorylation of the kinase did not appear to alter the overall rate of rhodopsin phosphorylation or the apparent KM (0.6 microM) for purified reconstituted rhodopsin. Peptides corresponding to sequences within opsin loops 3-4 and 5-6 and the COOH terminus inhibited kinase phosphorylation of bleached rhodopsin, suggesting at least three potential sites to account for the stable high affinity binding of rhodopsin kinase to the bleached photoreceptor molecule that are at least in part distinct from the substrate sites for phosphorylation. These sequences are similar to those proposed for receptor recognition of G-proteins and indicate that the domains involved in light-dependent binding of rhodopsin kinase and retinal G-protein are similar or overlapping.
Kelleher
D J
DJ
Department of Biochemistry, University of Massachusetts Medical School, Worcester 01655.
Johnson
G L
GL
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins; D051606:G-Protein-Coupled Receptor Kinase 1; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D010786:Photoreceptor Cells / Q000201:enzymology*; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000201:enzymology*; D013379:Substrate Specificity
D005136:Eye Proteins; D010455:Peptides; D011494:Protein Kinases; D051606:G-Protein-Coupled Receptor Kinase 1